Steady-state localization of transmembrane proteins of the trans Golgi network (TGN) is achieved by dynamic recycling between TGN and endosomal compartments. These transport pathways localize processing enzymes (e.g. Kex2 protease and furin) to their sites of action, effect lysosomal/vacuolar biogenesis and regulate levels of plasma membrane transporters and receptors. The fundamental importance of these pathways is underscored by their evolutionary conservation. The need to define TGN-endosomal transport pathways biochemically led us in the last grant period to begin to develop rapid, quantitative assays for cell-free reconstitution of these transport pathways. In this grant period, we will specifically focus on vesicular transport from the TGN to the late endosome, a key pathway both in lysosomal biogenesis and in processing protein localization that has not been reconstituted in any other system. Our overall goals will be to define mechanisms of vesicle budding, cargo sorting and regulation and vesicle scission in the formation of the clathrin coated vesicles that function in this step. Strong preliminary data, both published and unpublished, form the basis for the proposed work. The Specific Aims of this proposal are: 1. To determine rigorously whether there is a clathrin-coated vesicle intermediate in the reaction, to purify and characterize this intermediate and determine its molecular composition and to devise a vesicle budding assay that provides a direct assay for cargo recruitment. 2. To elucidate the roles of Gga1/2p versus AP1 adaptors in vesicle formation and cargo selection and determine how they are recruited to membranes. 3. To determine how cargo proteins are recruited to TGN-PVC transport vesicles and how they interact with adaptors or other components of the vesicle coat. [unreadable] [unreadable]